LightCycler 480 Instrument Quick Guide

LightCycler® 480 Instrument Quick Guide

For life science research use only.Not for use in diagnostic procedures.

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LightCycler® 480 Quick Guide Page i5125-00-1113

General Information

The contents of this document, including all graphics and photographs, are the property of Roche Diagnostics. No part may be reproduced or transmitted in any form or by any means, electronic or mechanical, for any purpose, without the express written permission of Roche Diagnostics.

Roche Diagnostics has made every reasonable effort to ensure that all the information contained in this manual is correct at the time of printing. However, Roche Diagnostics reserves the right to make any changes necessary without notice as part of ongoing product development.

This LightCyler® 480 Instrument Quick Guide was created by the Roche Diagnostics Corporation Commercial Education department. Direct questions or concerns regarding the contents of this document to:Roche Diagnostics Corporation Commercial Education Department 9115 Hague RoadP.O. Box 50457Indianapolis, IN 46250-0457USA

5125-00-1113

LIGHTCYCLER is a trademark of Roche. The technology for LightCycler® is licensed from Idaho Technologies, Inc. All other trademarks are the property of their respective owners.©2013, Roche Diagnostics.

Distribution in USA by: Roche Diagnostics, Indianapolis, IN This LightCycler® 480 Instrument Quick Guide is printed in the US.

Additional copies of this document can be obtained by sending a message to: [email protected]

Page ii LightCycler® 480 Quick Guide5125-00-1113

Revision History

Revisions to this document are provided by Roche Diagnostics when necessary. No part of this document may be reproduced in any form or by any means without prior written consent.

Publication Reference Number Date Revision Purpose5125-00-1113 November 2013 New Document Creation

LightCycler® 480 Quick Guide Page 15125-00-1113

LightCycler® 480 Instrument Basics

General Required Materials

Standard Laboratory Equipment

Product Name

Microwell Plate Centrifuge

Nuclease-free, aerosol-resistant pipette tips

Microcentrifuge and 1.5 mL microcentrifuge tubes

Micropipettes (P10 or P20, P200, P1000 or equivalent)

Disposables

Product Name Catalog Number Description

LightCycler® 480 Multi-well Plate 96-well - White 04 729 692 001 50 plates and sealing foils

LightCycler® 480 Multi-well Plate 384-well - White 04 729 749 001 50 plates and sealing foils

LightCycler® 480 Multi-well Plate 96-well - Clear 05 102 413 001 50 plates and sealing foils

LightCycler® 480 Multi-well Plate 384-well - Clear 05 102 430 001 50 plates and sealing foils

LightCycler® 480 Sealing Foil 04 729 757 001 50 sealing foils

Reagents

Reaction Type Product Name Catalog Number Description

Reverse Transcription with RNAse H activity

Transcriptor 1st Strand cDNA Synthesis Kit

04 379 012 001 For 50 (20 µL) reactions

SYBR Green Master LightCycler® 480 SYBR Green I Master

04 707 516 001 5 mL (5 x 20 µL)2x concentrated Master Mix for 250 (20 µL) reactions

High Resolution Melting

LightCycler® 480 High Resolution Melting Master

04 909 631 001 5 mL (5 x 1 mL)2x concentrated 25 mL MgCl2 stock solution for 500 (20 µL) reactions

Hybridization Probes LightCycler® 480 Genotyping Master

04 707 524 001 1.5 mL5x concentrated Master Mix for 250 (20 µL) reactions

Hydrolysis Probes TaqMan® Probes or Universal Probe Library (UPL)

LightCycler® 480 Probes Master

04 707 494 001 5 mL (5 x 1 mL)2x concentrated Master Mix for 250 (20 µL) reactions

LightCycler® 480 Control Kit 04 710 924 001 Kit for quantitative Real-Time PCR and genotyping reactions

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Getting Started1. Log on to the LightCycler® computer.

Username: Operator

Password: LC480

2. Start the LightCycler® 480 software by double-clicking the <LightCycler480> icon.

3. Enter the username and assigned password to log in to the LightCycler® 480 software.

First Time Login (new database)

Username: admin

Password: LightCycler480

Normal Login

Username: admin

Password: Roche480



4. The overview screen displays. This screen allows entry of a new experiment with or without use of a template for the conditions, or run a previously programed macro.



Software Navigation Overview

Exit the software

Logout of the current database

Switch to Overview menu

Display Navigator window

Save the current experiment

Export the current experiment to an “.ixo” file

Close the current experiment

Print the current screen

Display Tools window

Navigator window: displays the objects stored in the database as folders and files, allowing the user to work with objects in the database as well as import and export

Exporting as .ixo: creates a copy which can be imported to another computer or database

Tools window: allows user to change passwords, edit system settings, view database status and Error Log, define filter combination settings





New Experiment from Template

Create a New Experiment from TemplateTemplates provide a convenient way to speed up the process of creating an experiment. You can use Roche general templates for the different types of Real-time PCR reactions, as well as create your own templates based on the specific conditions of your experiments.

1. Click New Experiment from Template in the Overview window.

2. The New Experiment from Template window displays the templates that match the connected instrument.



3. Select a run template.

a. Dual Color Hydrolysis Probes/UPL Probe

• duplex reactions using Hydrolysis Probes (UPL/TaqMan) labeled with FAM and VIC, HEX or Yellow 555

b. End-point Genotyping (PCR read)

• genotyping with FAM and VIC or HEX-labeled Hydrolysis or TaqMan probes, PCR and data collection (Allelic discrimination)

c. End-point Genotyping (Pre-Post read)• genotyping with FAM and VIC or HEX-labeled probes, data collection only, PCR performed

in another instrument (Allelic Discrimination)

d. Gene Scanning

• basic protocol for High Resolution Melting experiments, including touch-down protocol

e. HybProbe

• reactions using Hybridization Probes, labeled with Fluorescein and LightCycler Red 640

f. Mono Color Hydrolysis Probe/UPL

• reactions with Hydrolysis Probes (UPL/TaqMan) labeled with FAM

g. Simple Probes

• melting curve genotyping reactions using a single Fluorescein-labeled probe (Simple Probe)

h. SYBR Green

• reactions using SYBR Green and simliar double-strand-intercalating dyes, includes melting curve

4. Click . The selected run-template is applied.

5. Modify run conditions (reaction volume, number of cycles, target temperature, hold, ramp rate, etc.) as needed.



6. The “Start Run” icon becomes active once the multi-well plate is loaded in the instrument.

7. Click Start Run.

8. Enter the experiment name in the dialog box.



Save Run Conditions as a TemplateIf you modify the run conditions, you can save it as a new template for future use.

1. Open or create the run to be used as a template.

2. Click the drop-down next to “Apply Template” and select Save as Template.

3. Name the run template and select a location to save the template.

Note: The default storage location for templates is the user’s Template folder.

4. Click . The template is saved and will appear in the New Experiment from Template window in the future.


Basic Real-Time Chemistry Protocols

Typical SYBR Green Reaction Protocol

Reaction Setup

Reagent Final Concentration Volume (µL) / one reactionWater 1.0Primer - Forward (5 µM Stock) 0.5 µM 2.0Primer - Reverse (5 µM Stock) 0.5 µM 2.0LightCycler® 480 SYBR Green I Master

1x 10.0

Total 15.0Add 5 µL template DNA for a total volume of 20 µL

Run ProgramProgram Name Cycles Analysis Mode

Pre-incubation 1 None

Target (°C) Acquisition Mode Hold (hh:mm:ss)95 None 00:05:00

Program Name Cycles Analysis Mode

Amplification 45 Quantification

Target (°C) Acquisition Mode Hold (hh:mm:ss)95 None 00:00:1060 None 00:00:1072 Single 00:00:10


Melting Curve 1 Melting Curves

Target (°C) Acquisition Mode Hold (hh:mm:ss) Acquisitions (per °C) 95 None 00:00:0565 None 00:01:0097 Continuous 5


Cooling 1 None




Typical Mono Color Hydrolysis Probe (TaqMan®/UPL) Reaction Protocol

Reaction Setup

Reagent Final Concentration Volume (µL) / one reactionWater 0.6Primer - Forward (5 µM Stock) 0.5 µM 2.0Primer - Reverse (5 µM Stock) 0.5 µM 2.0Probe (10 µM Stock) 0.2 µM 0.4LightCycler® 480 Probe Master 1x 10.0







Target (°C) Acquisition Mode Hold (hh:mm:ss)95 None 00:00:1060 Single 00:00:3072 None 00:00:01


Cooling 1 None




Typical Hybridization Probe (HybProbe) Reaction Protocol

Reaction Setup

Reagent Final Concentration Volume (µL) / one reactionWater 6.2Primer - Forward (5 µM Stock) 0.5 µM 2.0Primer - Reverse (5 µM Stock) 0.5 µM 2.0Fluorescein Probe (10 µM Stock) 0.2 µM 0.4Red Fluor Probe (10 µM Stock) 0.2 µM 0.4LightCycler® 480 Genotyping Master 1x 4.0







Target (°C) Acquisition Mode Hold (hh:mm:ss)95 None 00:00:1060 Single 00:00:1072 None 00:00:10


Melting Curve 1 Melting Curves

Target (°C) Acquisition Mode Hold (hh:mm:ss) Acquisitions (per °C) 95 None 00:01:0040 None 00:02:0095 Continuous 5


Cooling 1 None





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LightCycler 480 Instrument Quick Guide

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